Abstract
Hepatic stellate cells (HSCs) constitute liver sinusoids and have contractility, thereby regulating portal blood flow. Although it has been hypothesized that NO production by HSCs may also contribute to the regulation of portal circulation, there is no direct evidence for this novel function of HSCs, mainly because possible contamination of sinusoidal endothelial cells could not be avoided completely. Here we obtain the direct evidence of NO production in HSCs, using single-cell system andα-SMA as a marker of HSCs. Confocal images of immunostaining showed thatα-SMA-positive rat HSCs in primary culture expressed eNOS and caveolin-1, both of which were colocalized in the cell membrane at rest. In contrast, ATP and sphingomyelinase (SMase), which produces a lipid messenger, ceramide, induced the translocation of eNOS from caveolin-localizing cell membrane to the cytosol, as being compatible with the notion that eNOS activation requires its dissociation from caveolin, which binds and inhibits eNOS in the cell membrane at rest. Simultaneous fluorometric measurements of cytosolic Ca2+ concentration ([Ca2+]i) and NO production in single HSCs revealed that SMase induced NO production without [Ca2+]i elevation, whereas ATP elevated the both parameters. Western blotting showed the presence of eNOS, but not iNOS. Our results also indicate that the activation of eNOS in HSCs is mediated by not only a well-known Ca2dependent mechanism but also by a novel Ca2independent, probably ceramide-mediated, one. [Jpn J Physiol 54 Suppl:S252 (2004)]
